Protein kinase resource: an integrated environment for phosphorylation research

The protein kinase superfamily is an important group of enzymes controlling cellular signaling cascades. The increasing amount of available experimental data provides a foundation for deeper understanding of details of signaling systems and the underlying cellular processes. Here, we describe the Protein Kinase Resource, an integrated online service that provides access to information relevant to cell signaling and enables kinase researchers to visualize and analyze the data directly in an online environment. The data set is synchronized with Uniprot and Protein Data Bank (PDB) databases and is regularly updated and verified. Additional annotation includes interactive display of domain composition, cross-references between orthologs and functional mapping to OMIM records. The Protein Kinase Resource provides an integrated view of the protein kinase superfamily by linking data with their visual representation. Thus, human kinases can be mapped onto the human kinome tree via an interactive display. Sequence and structure data can be easily displayed using applications developed for the PKR and integrated with the website and the underlying database. Advanced search mechanisms, such as multiparameter lookup, sequence pattern, and blast search, enable fast access to the desired information, while statistics tools provide the ability to analyze the relationships among the kinases under study. The integration of data presentation and visualization implemented in the Protein Kinase Resource can be adapted by other online providers of scientific data and should become an effective way to access available experimental information.     

Meiotic localization of Mre11 and Rad50 in wild type, <Emphasis Type="Italic">spo11-1</Emphasis>, and MRN complex mutants of <Emphasis Type="Italic">Coprinus cinereus</Emphasis>

The Mre11-Rad50-Nbs1 (MRN) complex is required for numerous cellular processes that involve interactions with DNA double-strand breaks. For the majority of these processes, the MRN complex is thought to act as a unit, with each protein aiding the activity of the others. We have examined the relationship between Mre11 and Rad50 during meiosis in the basidiomycete Coprinus cinereus (Coprinopsis cinerea), investigating to what extent activities of Mre11 and Rad50 are interdependent. We showed that mre11-1 is epistatic to rad50-1 with respect to the time of meiotic arrest, indicating that Mre11 activity facilitates the diffuse diplotene arrest of rad50 mutants. Anti-Mre11 and anti-Rad50 antibodies were used to examine MRN complex localization in a wild-type strain and in spo11, mre11, and rad50 mutants. In wild type, numbers of Mre11 and Rad50 foci peaked at time points corresponding to leptotene and early zygotene. In the spo11-1 mutant, which is defective in meiotic double-strand break formation, foci accumulated throughout prophase I. Of seven MRN mutants examined, only two rad50 strains exhibited Mre11 and Rad50 foci that localized to chromatin, although Mre11 protein was found in the cell for all of them. Analysis of predicted mutant structures showed that stable localization of Mre11 and Rad50 does not depend upon a wild-type hook-proximal coiled coil, but does require the presence of the Rad50 ATPase/adenylate cyclase domains. We found that Mre11 and Rad50 were interdependent for binding to meiotic chromosomes. However, the majority of foci observed apparently contained only one of the two proteins. Independent Mre11 and Rad50 foci might indicate disassociation of the complex during meiosis or could reflect independent structural roles for the two proteins in meiotic chromatin. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The structures of frataxin oligomers reveal the mechanism for the delivery and detoxification of iron

Defects in the mitochondrial protein frataxin are responsible for Friedreich ataxia, a neurodegenerative and cardiac disease that affects 1:40,000 children. Here, we present the crystal structures of the iron-free and iron-loaded frataxin trimers, and a single-particle electron microscopy reconstruction of a 24 subunit oligomer. The structures reveal fundamental aspects of the frataxin mechanism. The trimer has a central channel in which one atom of iron binds. Two conformations of the channel with different metal-binding affinities suggest that a gating mechanism controls whether the bound iron is delivered to other proteins or transferred to detoxification sites. The trimer constitutes the basic structural unit of the 24 subunit oligomer. The architecture of this oligomer and several features of the trimer structure demonstrate striking similarities to the iron-storage protein ferritin. The data reveal how stepwise assembly provides frataxin with the structural flexibility to perform two functions: metal delivery and detoxification.     

Overexpression of human KCNA5 increases IK V and enhances apoptosis

Apoptotic cell shrinkage, an early hallmark of apoptosis, is regulated by K⁺ efflux and K⁺ channel activity. Inhibited apoptosis and downregulated K⁺ channels in pulmonary artery smooth muscle cells (PASMC) have been implicated in development of pulmonary vascular medial hypertrophy and pulmonary hypertension. The objective of this study was to test the hypothesis that overexpression of KCNA5, which encodes a delayed-rectifier voltage-gated K⁺ (Kv) channel, increases K⁺ currents and enhances apoptosis. Transient transfection of KCNA5 caused 25- to 34-fold increase in KCNA5 channel protein level and 24- to 29-fold increase in Kv channel current (I_K(V)) at +60 mV in COS-7 and rat PASMC, respectively. In KCNA5-transfected COS-7 cells, staurosporine (ST)-mediated increases in caspase-3 activity and the percentage of cells undergoing apoptosis were both enhanced, whereas basal apoptosis (without ST stimulation) was unchanged compared with cells transfected with an empty vector. In rat PASMC, however, transfection of KCNA5 alone caused marked increase in basal apoptosis, in addition to enhancing ST-mediated apoptosis. Furthermore, ST-induced apoptotic cell shrinkage was significantly accelerated in COS-7 cells and rat PASMC transfected with KCNA5, and blockade of KCNA5 channels with 4-aminopyridine (4-AP) reduced K⁺ currents through KCNA5 channels and inhibited ST-induced apoptosis in KCNA5-transfected COS-7 cells. Overexpression of the human KCNA5 gene increases K⁺ currents (i.e., K⁺ efflux or loss), accelerates apoptotic volume decrease (AVD), increases caspase-3 activity, and induces apoptosis. Induction of apoptosis in PASMC by KCNA5 gene transfer may serve as an important strategy for preventing the progression of pulmonary vascular wall thickening and for treating patients with idiopathic pulmonary arterial hypertension (IPAH). potassium ion channel; pulmonary hypertension     

Structural dynamics of myoglobin: an infrared kinetic study of ligand migration in mutants YQR and YQRF

Recombination of carbon monoxide to myoglobin mutants YQR and YQRF was studied using transient infrared absorption spectroscopy and Fourier transform infrared-temperature derivative spectroscopy (FTIR-TDS). Photoproduct states B, C', C" and D associated with ligands residing in different protein cavities have been identified. After photolysis, ligands migrate to primary docking site B and subsequently rebind or escape to a secondary site (C) within the Xe4 cavity. For YQR, a global analysis of the isothermal rebinding kinetics below 160 K and the TDS data reveal a correlation between the enthalpy barriers governing the two processes. Above 120 K, a protein conformational change in both YQR and YQRF converts photoproduct C' into C" with markedly slowed kinetics. Above approximately 180 K, ligands migrate to the proximal Xe1 site (D) and also exit into the solvent, from where they rebind in a bimolecular reaction.     

Mechanical model of the nucleosome and chromatin

A theoretical framework for evaluating the approximate energy and dynamic properties associated with the folding of DNA into nucleosomes and chromatin is presented. Experimentally determined elastic constants of linear DNA and a simple fold geometry are assumed in order to derive elastic constants for extended and condensed chromatin. The model predicts the Young s modulus of extended and condensed chromatin to within an order of magnitude of experimentally determined values. Thus we demonstrate that the elastic properties of DNA are a primary determinant of the elastic properties of the higher order folded states. The derived elastic constants are used to predict the speed of propagation of small amplitude waves that excite an extension(sound), twist, bend or shear motion in each folded state. Taken together the results demonstrate that folding creates a hierarchy of time, length and energy scales.     

Mitochondrial processing peptidases

Three peptidases are responsible for the proteolytic processing of both nuclearly and mitochondrially encoded precursor polypeptides targeted to the various subcompartments of the mitochondria. Mitochondrial processing peptidase (MPP) cleaves the vast majority of mitochondrial proteins, while inner membrane peptidase (IMP) and mitochondrial intermediate peptidase (MIP) process specific subsets of precursor polypeptides. All three enzymes are structurally and functionally conserved across species, and their human homologues begin to be recognized as potential players in mitochondrial disease.     

Raman spectroscopy of blood serum for Alzheimer's disease diagnostics: specificity relative to other types of dementia

The key moment for efficiently and accurately diagnosing dementia occurs during the early stages. This is particularly true for Alzheimer's disease (AD). In this proof‐of‐concept study, we applied near infrared (NIR) Raman microspectroscopy of blood serum together with advanced multivariate statistics for the selective identification of AD. We analyzed data from 20 AD patients, 18 patients with other neurodegenerative dementias (OD) and 10 healthy control (HC) subjects. NIR Raman microspectroscopy differentiated patients with more than 95% sensitivity and specificity. We demonstrated the high discriminative power of artificial neural network (ANN) classification models, thus revealing the high potential of this developed methodology for the differential diagnosis of AD. Raman spectroscopic, blood‐based tests may aid clinical assessments for the effective and accurate differential diagnosis of AD, decrease the labor, time and cost of diagnosis, and be useful for screening patient populations for AD development and progression.

Multivariate data analysis of blood serum Raman spectra allows for the differentiation between patients with Alzheimer's disease, other types of dementia and healthy individuals.     

Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits

Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca²⁺]_i in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1 μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca²⁺]_i by about 70 %. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2 μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9415-6) contains supplementary material, which is available to authorized users.